Capturing Structural Variants of Herpes Simplex Virus Genome in Full Length by Oxford Nanopore Sequencing

ABSTRACT Genome sequencing and assembly of viral genomes within the Herpesviridae family, particularly herpes simplex virus (HSV), have been challenging due to the large size (~154 Kb), high GC content (68%), and nucleotide variations arising during replication. Oxford Nanopore Technology (ONT) has been successful in obtaining read lengths ranging from 100 Kb up to 2.3 Mb. We have optimized DNA extraction and sequencing with ONT to capture the whole genome of HSV-1 as a single read. Although previous studies described the presence of four different genome isomers of HSV, we provided the first report on capturing all four variants’ full-length genome as single reads. These isomers were found to be present in almost equal proportion in the sequenced DNA preparation. IMPORTANCE With the advent of next-generation sequencing platforms, genome sequencing of viruses can be performed in a relatively shorter time frame in even the most austere conditions. Ultralong read sequencing platforms, such as Oxford Nanopore Technology (ONT), have made it possible to capture the full-length genome of DNA viruses as a single read. By optimizing ONT for this purpose, we captured the genome (~154 Kb) of a clinical strain of herpes simplex virus 1 (HSV-1). Additionally, we captured full-length sequences of the four isomers of lab-grown HSV-1 virus and were able to determine the frequency of each within the isogenic population. This method will open new directions in studying the significance of these isomers and their clinical relevance to HSV-1 infections. It will also improve basic studies on the recombination and replication of this virus.

I thank you for your contribution to those interested in herpesviruses sequencing. Two experts in the field have reviewed this manuscript, and the comments were split on the novelty and significance of the findings. I've also examined the submission and concluded that the work is technically sound and within the scope of Spectrum's mission to publish Observations that are rigorous without consideration of potential impact.
I look forward to a thoughtful revision that includes data sharing depositions and a more thorough review of similar previously published methods.
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Reviewer #1 (Comments for the Author): This is a very interesting study, the full-genome sequences of the virus are extremely important. The manuscript could gain by citing and commenting on a previous study performed by Karamitros et al. where Nanopore and short read sequenced were combined. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910999/ Reviewer #2 (Public repository details (Required)): ONT sequencing data and Illumina sequencing data need to be deposited to NCBI SRA database Reviewer #2 (Comments for the Author): In "Capturing structural variants of herpes simplex virus genome in full length by Oxford Nanopore sequencing", authors improved HSV-1 improved viral genome DNA, observed 15 full-length genome of four types isomers and obtained the composition of four isomers based on reads containing IRL, IRS and flanking regions.
Both the viral DNA prep method improvement and virology discovery aren't significant enough for publication.

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Thank you for submitting your paper to Microbiology Spectrum.
In "Capturing structural variants of herpes simplex virus genome in full length by Oxford Nanopore sequencing", authors improved HSV-1 improved viral genome DNA, observed 15 full-length genomes of four types of isomers and obtained the composition of four isomers based on reads containing IRL, IRS and flanking regions.
Both the viral DNA prep method improvement and virology discovery aren't significant enough for publication. We thank you and the two reviewers for taking the time to review our submission and their comments. Please find below the pointwise response in bold letters. The manuscript has been revised and we have highlighted all changes in the manuscript.

Reviewer #1
The raw sequencing data need to be deposited to the Sequencing Read Archive.

We submitted both ONT and Illumina Miseq raw data to NCBI-SRA database and included the SRA number in the revised manuscript
This is a very interesting study, the full-genome sequences of the virus are extremely important. The manuscript could gain by citing and commenting on a previous study performed by Karamitros et al. where Nanopore and short read sequenced were combined. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910999/ We thank the reviewer for pointing out this important study. We discussed the findings of this publication and included in the revised manuscript

Reviewer #2
ONT sequencing data and Illumina sequencing data need to be deposited to NCBI SRA database

We submitted both ONT and Illumina Miseq raw data to NCBI-SRA database and included the SRA number in the revised manuscript
In "Capturing structural variants of herpes simplex virus genome in full length by Oxford Nanopore sequencing", authors improved HSV-1 improved viral genome DNA, observed 15 full-length genome of four types isomers and obtained the composition of four isomers based on reads containing IRL, IRS and flanking regions.
Both the viral DNA prep method improvement and virology discovery aren't significant enough for publication.
To our knowledge, this would be first report on capturing full-length sequences of the four isomers as single read from a herpesvirus DNA preparation. This is important as mutations are often found among the genes in inverted repeat elements, which are present in two copies and hard to capture with shotgun sequencing approaches. Causality of mutation-induced phenotypes could only be concluded with full-length genome sequences. We believe that future work will improve using this DNA isolation method and ONT would be the assay for studying HSV. B 3 x1.1 is a highly virulent recent clinical isolate that is a valuable strain for future vaccine works and genetic studies.
We thank the reviewers for the opportunity to improve our manuscript, William R. Jacobs Jr, PhD Thank you for your contribution of methods that may further the study of herpesvirus genomics.
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ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.